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Etest synergy testing of clinical isolates of Staphylococcus aureus demonstrating heterogeneous resistance to vancomycin
Diagnostic Microbiology and Infectious Disease 54 (2006) 73 – 77 www.elsevier.com/locate/diagmicrobio
Etest synergy testing of clinical isolates of Staphylococcus aureus demonstrating heterogeneous resistance to vancomycinB Brian T. Tsujia,b,1, Michael J. Rybaka,b,c,T a
Anti-Infective Research Laboratory, Department of Pharmacy Practice, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201, USA b Department of Pharmacy Services, Detroit Receiving Hospital and University Health Center, Detroit, MI 48201, USA c School of Medicine, Wayne State University, Detroit, MI 48201, USA Received 14 June 2005; revised 26 August 2005; accepted 29 August 2005
Abstract In search for potential synergistic antimicrobial combinations against Staphylococcus aureus isolates, which display heterogeneous resistance to vancomycin, we evaluated the activities of 21 various combinations involving ampicillin/sulbactam, daptomycin, gentamicin, linezolid, quinupristin/dalfopristin, rifampin, and vancomycin by Etest and time-kill methods. A number of combinations demonstrated either synergistic or additive effects against hGISA SA118 and GISA SA179. Agreement between the Etest and time-kill methods for detecting antimicrobial synergy ranged from 66.6% to 71.4%. The Etest method appears promising, and further investigations are warranted. D 2006 Elsevier Inc. All rights reserved. Keywords: Etest; Synergy heterogeneous resistant S. aureus; Daptomycin; Linezolid; Quinupristin/dalfopristin; Rifampin; Vancomycin Ampicillin/sulbactam; Gentamicin
1. Introduction Although the clinical significance of heterogeneous glycopeptide-intermediate Staphylococcus aureus (hGISA) infections has been debated and conflicting results concerning the prevalence of such isolates have been reported, S. aureus stains, which exhibit reduced susceptibility to vancomycin, continue to be a therapeutic problem (Ariza et al., 1999; Eguia et al., 2005; Fridkin et al., 2003; Hiramatsu et al., 1997; Howden et al., 2004; Liu and Chambers, 2003; Moore et al., 2003). Limited data exist on the utility of combination antimicrobial therapy against GISA and hGISA infections.
B A portion of this work was presented at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, October 2004. T Corresponding author. Anti-Infective Research Laboratory, Department of Pharmacy Practice 4148, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Avenue, Detroit, MI 48201. Tel.: +1-313-993-4673; fax: +1-313-577-8915. E-mail address: [email protected] (M.J. Rybak). 1 Current address: School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, Buffalo, NY 14260, USA.
0732-8893/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2005.08.014
The Epsilometer (Etest) is an agar diffusion method that quantitatively determines antimicrobial susceptibility. Etest has also been used to assess the synergistic activities of antimicrobial combinations against a wide variety resistant of organisms (White et al., 1996; Bonapace et al., 2000; Lewis et al., 2002). We used the Etest method to assess the activity of numerous antimicrobial combinations against GISA and hGISA and compared these results with timekill experiments. 2. Methods Two recently characterized S. aureus isolates that display heterogeneous resistance to vancomycin were evaluated (Rybak et al., 2005). Both isolates were obtained from patients with infective endocarditis: SA 118, which is a methicillin-susceptible isolate, and SA 179, which is methicillin-resistant isolate. Etest strips of ampicillin/sulbactam, gentamicin, linezolid, quinupristin/dalfopristin, rifampin, and vancomycin (AB Biodisk, Solna, Sweden) were used. Etest strips of daptomycin (Cubist Pharmaceuticals, Lexington, MA) were obtained from its respective manufacturer. Analytical
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B.T. Tsuji, M.J. Rybak / Diagnostic Microbiology and Infectious Disease 54 (2006) 73 – 77
Table 1 Antimicrobial susceptibilities (MIC in Ag/mL) Antimicrobial agent
hGISA Microdilution
Ampicillin/sulbactam Daptomycin Linezolid Gentamicin Quinupristin/ dalfopristin Rifampin Vancomycin
2 0.5 2 2 0.5 512 4
GISA Etest 1 0.5 2 2 0.5 N 32 4
Microdilution
Etest
8 0.5 1 2 0.5
4 0.5 1 2 0.5
0.25 8
0.16 8
grade powder of daptomycin (Cubist Pharmaceuticals, Lexington, MA) and quinupristin/dalfopristin (Aventis Pharmaceuticals, Colgate, PA) were obtained from their respective manufacturers. Ampicillin/sulbactam (Pfizer, Kalamazoo, MI), linezolid (Pfizer), gentamicin (Sigma, St. Louis, MO), rifampin (Sigma), and vancomycin (Sigma) were obtained commercially. Mueller–Hinton broth (Difco Laboratories, Detroit, MI) supplemented with calcium (25 mg/L) and magnesium (12.5 mg/L) was used for all broth microdilution susceptibility testing and time-kill experiments except daptomycin. Supplemented Mueller–Hinton broth titrated to physiological concentrations of calcium were used for all experiments involving daptomycin due to its dependency on calcium for its activity (Lamp and Rybak, 1993; Hanberger et al., 1991). Brain–heart infusion (BHI) agar was used for Etest susceptibility and synergy testing and quantification of timekill experiments. Isosensitest agar supplemented to physio-
logical concentrations of ionized calcium was used for Etest susceptibility and synergy testing involving daptomycin. MICs were determined by broth microdilution and Etest (see Table 1 for results). For broth microdilution, MICs were determined in triplicate in accordance with the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) guidelines. (NCCLS, 2005). Determination of MICs by Etest was preformed in triplicate according to the manufacturer’s recommendations. BHI agar plates were inoculated with a suspension of a 0.5 McFarland of test organism. One Etest strip was placed onto each BHI plate. The plates were incubated for 18–24 h at 35 8C. The inoculum and BHI agar plates were prepared as previously described. To test the combination of 2 antimicrobials, an Etest strip of the first antimicrobial was placed on an agar plate, as described previously (Bolstrum et al., 1989). Briefly, after 1 h of incubation at 35 8C, the first Etest strip was removed, and an Etest strip of the second antimicrobial was superimposed so that the respective MICs were aligned. The Etest method was preformed in duplicate for all organisms and combinations. The plates were then incubated 18 to 24 h at 35 8C. After incubation, the zones of inhibition were read. To interpret the combinations,Pthe cumulative fractional inhibitory concentration index ( FIC) was calculated for each antimicrobial combination as the sum of the individual FICs. The nature of interaction between 2 antibiotics (synergy, indifference, or antagonism) was determined based on FIC index: V 0.5 was considered synergy, N 0.5
Table 2 Etest versus time-kill method of detecting synergy Antimicrobial combination
hGISA SA118
GISA SA179
FIC index
Effect
Dlog10 CFU/mL
Effect
FIC index
Effect
Dlog10 CFU/mL
Effect
D + AS D + LZ D + QD D+V G + AS G+D G + LZ G + QD G+V LZ + AS QD + AS QD + LZ R + AS R+D R+G R + LZ R + QD R+V V + AS V + LZ V + QD
1.1 1.1 1.2 0.6 0.7 0.7 1.4 0.7 0.9 1.4 1.4 1.1 1.5 1.8 1.4 1.8 2.3 1.8 0.6 0.6 1.1
IND IND IND ADD ADD ADD IND ADD ADD IND IND IND IND IND IND IND IND IND ADD ADD IND
0.10 0.23 0.25 0.31 3.58 1.90 0.64 1.49 2.12 0.12 0.07 0.28 0.24 0.29 0.15 0.30 0.59 0.15 0.71 0.11 4.00
IND IND IND IND SYN ADD IND ADD SYN IND IND IND IND IND IND IND IND IND IND IND SYN
1.7 1.3 1.1 1.2 3.0 0.8 1.4 0.9 0.8 1.8 1.5 1.8 1.1 1.7 0.4 1.1 1.3 1.7 0.4 1.9 0.8
IND IND IND IND IND ADD IND ADD ADD IND IND IND IND IND SYN IND IND IND SYN IND ADD
0.16 0.09 0.07 0.04 1.00 0.12 1.66 1.38 3.48 0.01 0.16 0.05 5.00 0.04 3.28 0.66 0.91 0.47 0.36 0.29 1.24
IND IND IND IND ADD IND ADD ADD SYN IND IND IND SYN IND SYN IND IND IND IND IND ADD
Etest
Time-kill
Etest
Time-kill
SYN = synergy; ADD = additivity; IND = indifference; Dlog10 CFU/mL = change in log10 CFU/mL at 24 h as compared with the most active agent; AS = ampicillin/sulbactam; D = daptomycin; G = gentamicin; L = linezolid; QD = quinupristin/dalfopristin; R = rifampin; V = vancomycin.
B.T. Tsuji, M.J. Rybak / Diagnostic Microbiology and Infectious Disease 54 (2006) 73 – 77
75
Fig. 1. Selective synergistic and additive time-kill experiments against SA118 hGISA.
to V 1 was considered additivity, N1 to V 4 was considered indifference, and N4 was considered antagonism. The time-kill method of synergy testing was performed by the broth macrodilution technique in accordance with NCCLS guidelines (NCCLS, 2005) as previously described (Rybak et al., 2005) Each organism was tested against each antimicrobial agent alone and in combination. All antimicrobial agents were tested at 0.5 times their respective MIC. Synergy was defined as an increase in kill of z 2-log10 CFU/mL by combination of antimicrobials versus the most active single agent of that combination at 24 h. Additivity was defined as a 1- to 2-log10 CFU/mL increase in kill in comparison to the most active single agent. Combinations that resulted in z 1-log10 bacterial growth in comparison to the least active single agent were considered to represent antagonism. 3. Results Etest synergy results are reported in Table 2. The combinations that demonstrated additivity against SA 118 were daptomycin + vancomycin, gentamicin + ampicillin/
sulbactam, gentamicin + daptomycin, gentamicin + vancomycin, vancomycin + ampicillin/sulbactam, and vancomycin + linezolid according to Etest methods. Against SA 179, gentamicin + daptomycin, gentamicin + quinupristin/dalfopristin, gentamicin + vancomycin, and vancomycin + quinuprisin–dalfopristin demonstrated additivity according to Etest methods. In addition, rifampin + gentamicin and vancomycin + ampicillin/sulbactam resulted in synergy against SA 179 according to Etest methods. No antagonism was detected among both isolates. Time-kill experiments are listed in Figs. 1 and 2. Alone, all agents did not possess significant activity against both strains of SA 118 and SA 179, as they were tested at 0.5 MIC. For SA 118, the combinations that demonstrated additivity were gentamicin + daptomycin and gentamicin + quinupristin-dalfopristin. Gentamicin + ampicillin-sulbactam, gentamicin + vancomycin, and vancomycin + quinupristin/dalfopristin demonstrated synergy against SA 118 according to time-kill methods. Against SA 179, the combinations that demonstrated additivity were gentamicin + ampicillin-sulbactam, gentamicin + linezolid, gentamicin + quinupristin-dalfopristin,
Log10 CFU/ml
10
8
growth control vancomycin, ampicillin/sulbactam, quinupristin/dalfopristin, rifampin linezolid, gentamicin gentamicin + ampicillin/sulbactam
6
vancomycin + quinupristin/dalfopristin gentamicin + quinupristin/dalfopristin gentamicin + linezolid*
4
rifampin + gentamicin* gentamicin + vancomycin* rifampin + ampicillin/sulbactam*
2 0
8
16
24
Time (hour)
* = P < .05 Fig. 2. Select synergistic and additive time-kill experiments against SA179 GISA.
76
B.T. Tsuji, M.J. Rybak / Diagnostic Microbiology and Infectious Disease 54 (2006) 73 – 77
and vancomycin+quinupristin-dalfopristin. In addition, gentamicin + vancomycin, rifampin + ampicillin/sulbactam, and rifampin + gentamicin resulted in synergy against SA 179 according to time-kill methods. When comparing both methods, agreement between Etest and time-kill methods for detecting antimicrobial synergy was 66.6% for hVISA and 71.4% for GISA.
susceptibility to vancomycin should be preformed to confirm these findings. Caution is warranted as additional investigations are necessary in humans before these results are applied to clinical practice.
Acknowledgments We would like to thank Anne Bolstrum and Irene Miloff.
4. Discussion In the face of mounting antimicrobial resistance, the need to evaluate new therapeutic options against S. aureus exist. Combination therapy, using agents that together achieve synergistic or additive activity is one potential means to optimize the pharmacokinetic and pharmacodynamic properties of new and existing antimicrobials. In the present investigation, we examined the potential effectiveness of numerous antimicrobial combinations against 2 S. aureus isolates from 1987 and 1989, which display heterogeneous resistance to vancomycin by utilizing Etest and time-kill methods to evaluate synergy. We were encouraged by the activity of a number of combinations that demonstrated either for synergistic or additive effects against hGISA and GISA. Among the 21 combinations that we tested against hGISA, numerous combinations with gentamicin significantly resulted in synergy or additivity. In addition, we evaluated the utility of the Etest method to detect antimicrobial synergy and compared these results to time-kill experiments. Although each method evaluates different end points, we found 66.6% to 71.4% agreement. These results are similar to a previously published comparison of the checkerboard, time-kill, and Etest methods (White et al., 1996). Other studies have found similar favorable agreement when comparing the Etest to time-kill methods. (Bonapace et al., 2002; Manno et al., 2003; Orhan et al., 2005; Pankey et al., 2002). The Etest method has significant advantages over traditional methods of synergy detection including being less labor-intensive and easier to perform, which may be of utility in the clinical microbiology laboratory. However, although it is practical method compared with time-kill experiments, the Etest method does not provide a quantitative measure of bacterial density in evaluating antimicrobial activity. In addition, compared with other traditional methods such as the checkerboard method, although both methods use the FIC index to evaluate synergy, Etest methods are not based on conventional microdilution MIC data, which use 2-fold dilutions. Therefore, it is important to take these considerations into account when interpreting synergy data generated from Etest methodology. Although we found good agreement between both methods, we recognize that depending on the methodology used, results often vary, which has been demonstrated in other studies (Bonapace et al., 2002; Cappelletty and Rybak, 1996; White et al., 1996). Further investigations in other S. aureus strains that display reduced
References Ariza J, Pujol M, Cabo J, Pena C, Fernandez N, Linares J, Avats J, Gudiol F (1999) Vancomycin in surgical infections due to methicillin-resistant Staphylococcus aureus with heterogeneous resistance to vancomycin. Lancet 353:1587 – 1588. Bolstrum A, Arvdvison S, Ericson M, Karlsonn A (1989) New in vitro models for studying antibiotic combination using predefined antibiotics gradient. Progam and abstracts of the 16th International Congress of Chemotherapy, Jerusalem, Abstract No. 11. Bonapace CR, White RL, Friedrich LV, Bosso JA (2000) Evaluation of antibiotic synergy against Acinetobacter baumannii: a comparison with Etest, time-kill, and checkerboard methods. Diagn Microbiol Infect Dis 38:43 – 50. Bonapace CR, Bosso JA, Friedrich LV, White RL (2002) Comparison of methods of interpretation of checkerboard synergy testing. Diagn Microbiol Infect Dis 44:363 – 366. Cappelletty DM, Rybak MJ (1996) Comparison of methodologies for synergism testing of drug combinations against resistant strains of Pseudomonas aeruginosa. Antimicrob Agents Chemother 40:677 – 683. Eguia JM, Liu C, Moore M, Wrone EM, Pont J, Gerberding JL, Chambers HF (2005) Low colonization prevalence of Staphylococcus aureus with reduced vancomycin susceptibility among patients undergoing hemodialysis in the San Francisco Bay area. Clin Infect Dis 1617 – 1624. Fridkin SK, Hageman J, McDougal LK, Mohammed J, Jarvis WR, Perl TM, Tenover FC (2003) Epidemiological and microbiological characterization of infections caused by Staphylococcus aureus with reduced susceptibility to vancomycin, United States, 1997–2001. Clin Infect Dis 429 – 439. Hiramatsu K, Aritaka N, Hanaki H, Kawasaki S, Hosoda Y, Hori S, et al. (1997) Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet 350:70 – 73. Howden BP, Ward PB, Charles PGP, Korman TM, Fuller A, du Cros P, Grabsch EA, Roberts SA, Robson J, Read K, Bak N, Hurley J, Johnson PDR, Morris AJ, Mayall BC, Grayson ML (2004) Treatment outcomes for serious infections caused by methicillin-resistant Staphylococcus aureus with reduced vancomycin susceptibility. Clin Infect Dis 38:521 – 528. Hanberger H, Nilsson LE, Maller R, Isaksson B (1991) Pharmacodynamics of daptomycin and vancomycin on Enterococcus faecalis and Staphylococcus aureus demonstrated by studies of initial killing and postantibiotic effect and influence of Ca2+ and albumin on these drugs. Antimicrob Agents Chemother 35:1710 – 1716. Lamp KC, Rybak MJ (1993) Teicoplanin and daptomycin bactericidal activities in the presence of albumin or serum under controlled conditions of pH and ionized calcium. Antimicrob Agents Chemother 37:605 – 609. Lewis RE, Diekema DJ, Messer SA, Pfaller MA, Klepser ME (2002) Comparison of Etest, chequerboard dilution and time-kill studies for the detection of synergy or antagonism between antifungal agents tested against Candida species. J Antimicrob Chemother 345 – 351. Liu C, Chambers HF (2003) Staphylococcus aureus with heterogeneous resistance to vancomycin: epidemiology, clinical significance, and critical assessment of diagnostic methods. Antimicrob Agents Chemother 47:3040 – 3045. Manno G, Ugolotti E, Belli ML, Fenu ML, Romano L, Cruciani M (2003) Use of the E test to assess synergy of antibiotic combinations against
B.T. Tsuji, M.J. Rybak / Diagnostic Microbiology and Infectious Disease 54 (2006) 73 – 77 isolates of Burkholderia cepacia-complex from patients with cystic fibrosis. Eur J Clin Microbiol Infect Dis 28 – 34. Moore MR, Perdreau-Remington F, Chambers HF (2003) Vancomycin treatment failure associated with heterogeneous vancomycin-intermediate Staphylococcus aureus in a patient with endocarditis and in the rabbit model of endocarditis. Antimicrob Agents Chemother 47:1262 – 1266. National Committee for Clinical Laboratory Standards (2005) Performance standard for antimicrobial susceptibility testing Document M100-S15. Wayne (PA)7 NCCLS. Orhan G, Bayram A, Zer Y, Balci I (2005) Synergy tests by E test and checkerboard methods of antimicrobial combinations against Brucella melitensis. J Clin Microbiol 140 – 143.
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Pankey GA, Ashcraft D, Prakash O (2002) Etest synergy testing of oxacillin-resistant Staphylococcus aureus (ORSA) with linezolid (L) and moxifloxacin (M) (Abstract: E-1133). 42nd ICAAC Abstracts, American Society for Microbiology, September 27–30, 2002, San Diego, CA. (158). Rybak MJ, Cha R, Cheung CM, Meka VG, Kaatz GW (2005) Clinical isolates of Staphylococcus aureus from 1987 and 1989 demonstrating heterogeneous resistance to vancomycin and teicoplanin. Diagn Microbiol Infect Dis 119 – 125. White RL, Burgess DS, Manduru M, Bosso JA (1996) Comparison of three different in vitro methods of detecting synergy: time-kill, checkerboard, and E test. Antimicrob Agents Chemother 40:1914 – 1918.
Etest synergy testing of clinical isolates of Staphylococcus aureus demonstrating heterogeneous resistance to vancomycinB Brian T. Tsujia,b,1, Michael J. Rybaka,b,c,T a
Anti-Infective Research Laboratory, Department of Pharmacy Practice, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201, USA b Department of Pharmacy Services, Detroit Receiving Hospital and University Health Center, Detroit, MI 48201, USA c School of Medicine, Wayne State University, Detroit, MI 48201, USA Received 14 June 2005; revised 26 August 2005; accepted 29 August 2005
Abstract In search for potential synergistic antimicrobial combinations against Staphylococcus aureus isolates, which display heterogeneous resistance to vancomycin, we evaluated the activities of 21 various combinations involving ampicillin/sulbactam, daptomycin, gentamicin, linezolid, quinupristin/dalfopristin, rifampin, and vancomycin by Etest and time-kill methods. A number of combinations demonstrated either synergistic or additive effects against hGISA SA118 and GISA SA179. Agreement between the Etest and time-kill methods for detecting antimicrobial synergy ranged from 66.6% to 71.4%. The Etest method appears promising, and further investigations are warranted. D 2006 Elsevier Inc. All rights reserved. Keywords: Etest; Synergy heterogeneous resistant S. aureus; Daptomycin; Linezolid; Quinupristin/dalfopristin; Rifampin; Vancomycin Ampicillin/sulbactam; Gentamicin
1. Introduction Although the clinical significance of heterogeneous glycopeptide-intermediate Staphylococcus aureus (hGISA) infections has been debated and conflicting results concerning the prevalence of such isolates have been reported, S. aureus stains, which exhibit reduced susceptibility to vancomycin, continue to be a therapeutic problem (Ariza et al., 1999; Eguia et al., 2005; Fridkin et al., 2003; Hiramatsu et al., 1997; Howden et al., 2004; Liu and Chambers, 2003; Moore et al., 2003). Limited data exist on the utility of combination antimicrobial therapy against GISA and hGISA infections.
B A portion of this work was presented at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, October 2004. T Corresponding author. Anti-Infective Research Laboratory, Department of Pharmacy Practice 4148, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Avenue, Detroit, MI 48201. Tel.: +1-313-993-4673; fax: +1-313-577-8915. E-mail address: [email protected] (M.J. Rybak). 1 Current address: School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, Buffalo, NY 14260, USA.
0732-8893/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2005.08.014
The Epsilometer (Etest) is an agar diffusion method that quantitatively determines antimicrobial susceptibility. Etest has also been used to assess the synergistic activities of antimicrobial combinations against a wide variety resistant of organisms (White et al., 1996; Bonapace et al., 2000; Lewis et al., 2002). We used the Etest method to assess the activity of numerous antimicrobial combinations against GISA and hGISA and compared these results with timekill experiments. 2. Methods Two recently characterized S. aureus isolates that display heterogeneous resistance to vancomycin were evaluated (Rybak et al., 2005). Both isolates were obtained from patients with infective endocarditis: SA 118, which is a methicillin-susceptible isolate, and SA 179, which is methicillin-resistant isolate. Etest strips of ampicillin/sulbactam, gentamicin, linezolid, quinupristin/dalfopristin, rifampin, and vancomycin (AB Biodisk, Solna, Sweden) were used. Etest strips of daptomycin (Cubist Pharmaceuticals, Lexington, MA) were obtained from its respective manufacturer. Analytical
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B.T. Tsuji, M.J. Rybak / Diagnostic Microbiology and Infectious Disease 54 (2006) 73 – 77
Table 1 Antimicrobial susceptibilities (MIC in Ag/mL) Antimicrobial agent
hGISA Microdilution
Ampicillin/sulbactam Daptomycin Linezolid Gentamicin Quinupristin/ dalfopristin Rifampin Vancomycin
2 0.5 2 2 0.5 512 4
GISA Etest 1 0.5 2 2 0.5 N 32 4
Microdilution
Etest
8 0.5 1 2 0.5
4 0.5 1 2 0.5
0.25 8
0.16 8
grade powder of daptomycin (Cubist Pharmaceuticals, Lexington, MA) and quinupristin/dalfopristin (Aventis Pharmaceuticals, Colgate, PA) were obtained from their respective manufacturers. Ampicillin/sulbactam (Pfizer, Kalamazoo, MI), linezolid (Pfizer), gentamicin (Sigma, St. Louis, MO), rifampin (Sigma), and vancomycin (Sigma) were obtained commercially. Mueller–Hinton broth (Difco Laboratories, Detroit, MI) supplemented with calcium (25 mg/L) and magnesium (12.5 mg/L) was used for all broth microdilution susceptibility testing and time-kill experiments except daptomycin. Supplemented Mueller–Hinton broth titrated to physiological concentrations of calcium were used for all experiments involving daptomycin due to its dependency on calcium for its activity (Lamp and Rybak, 1993; Hanberger et al., 1991). Brain–heart infusion (BHI) agar was used for Etest susceptibility and synergy testing and quantification of timekill experiments. Isosensitest agar supplemented to physio-
logical concentrations of ionized calcium was used for Etest susceptibility and synergy testing involving daptomycin. MICs were determined by broth microdilution and Etest (see Table 1 for results). For broth microdilution, MICs were determined in triplicate in accordance with the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) guidelines. (NCCLS, 2005). Determination of MICs by Etest was preformed in triplicate according to the manufacturer’s recommendations. BHI agar plates were inoculated with a suspension of a 0.5 McFarland of test organism. One Etest strip was placed onto each BHI plate. The plates were incubated for 18–24 h at 35 8C. The inoculum and BHI agar plates were prepared as previously described. To test the combination of 2 antimicrobials, an Etest strip of the first antimicrobial was placed on an agar plate, as described previously (Bolstrum et al., 1989). Briefly, after 1 h of incubation at 35 8C, the first Etest strip was removed, and an Etest strip of the second antimicrobial was superimposed so that the respective MICs were aligned. The Etest method was preformed in duplicate for all organisms and combinations. The plates were then incubated 18 to 24 h at 35 8C. After incubation, the zones of inhibition were read. To interpret the combinations,Pthe cumulative fractional inhibitory concentration index ( FIC) was calculated for each antimicrobial combination as the sum of the individual FICs. The nature of interaction between 2 antibiotics (synergy, indifference, or antagonism) was determined based on FIC index: V 0.5 was considered synergy, N 0.5
Table 2 Etest versus time-kill method of detecting synergy Antimicrobial combination
hGISA SA118
GISA SA179
FIC index
Effect
Dlog10 CFU/mL
Effect
FIC index
Effect
Dlog10 CFU/mL
Effect
D + AS D + LZ D + QD D+V G + AS G+D G + LZ G + QD G+V LZ + AS QD + AS QD + LZ R + AS R+D R+G R + LZ R + QD R+V V + AS V + LZ V + QD
1.1 1.1 1.2 0.6 0.7 0.7 1.4 0.7 0.9 1.4 1.4 1.1 1.5 1.8 1.4 1.8 2.3 1.8 0.6 0.6 1.1
IND IND IND ADD ADD ADD IND ADD ADD IND IND IND IND IND IND IND IND IND ADD ADD IND
0.10 0.23 0.25 0.31 3.58 1.90 0.64 1.49 2.12 0.12 0.07 0.28 0.24 0.29 0.15 0.30 0.59 0.15 0.71 0.11 4.00
IND IND IND IND SYN ADD IND ADD SYN IND IND IND IND IND IND IND IND IND IND IND SYN
1.7 1.3 1.1 1.2 3.0 0.8 1.4 0.9 0.8 1.8 1.5 1.8 1.1 1.7 0.4 1.1 1.3 1.7 0.4 1.9 0.8
IND IND IND IND IND ADD IND ADD ADD IND IND IND IND IND SYN IND IND IND SYN IND ADD
0.16 0.09 0.07 0.04 1.00 0.12 1.66 1.38 3.48 0.01 0.16 0.05 5.00 0.04 3.28 0.66 0.91 0.47 0.36 0.29 1.24
IND IND IND IND ADD IND ADD ADD SYN IND IND IND SYN IND SYN IND IND IND IND IND ADD
Etest
Time-kill
Etest
Time-kill
SYN = synergy; ADD = additivity; IND = indifference; Dlog10 CFU/mL = change in log10 CFU/mL at 24 h as compared with the most active agent; AS = ampicillin/sulbactam; D = daptomycin; G = gentamicin; L = linezolid; QD = quinupristin/dalfopristin; R = rifampin; V = vancomycin.
B.T. Tsuji, M.J. Rybak / Diagnostic Microbiology and Infectious Disease 54 (2006) 73 – 77
75
Fig. 1. Selective synergistic and additive time-kill experiments against SA118 hGISA.
to V 1 was considered additivity, N1 to V 4 was considered indifference, and N4 was considered antagonism. The time-kill method of synergy testing was performed by the broth macrodilution technique in accordance with NCCLS guidelines (NCCLS, 2005) as previously described (Rybak et al., 2005) Each organism was tested against each antimicrobial agent alone and in combination. All antimicrobial agents were tested at 0.5 times their respective MIC. Synergy was defined as an increase in kill of z 2-log10 CFU/mL by combination of antimicrobials versus the most active single agent of that combination at 24 h. Additivity was defined as a 1- to 2-log10 CFU/mL increase in kill in comparison to the most active single agent. Combinations that resulted in z 1-log10 bacterial growth in comparison to the least active single agent were considered to represent antagonism. 3. Results Etest synergy results are reported in Table 2. The combinations that demonstrated additivity against SA 118 were daptomycin + vancomycin, gentamicin + ampicillin/
sulbactam, gentamicin + daptomycin, gentamicin + vancomycin, vancomycin + ampicillin/sulbactam, and vancomycin + linezolid according to Etest methods. Against SA 179, gentamicin + daptomycin, gentamicin + quinupristin/dalfopristin, gentamicin + vancomycin, and vancomycin + quinuprisin–dalfopristin demonstrated additivity according to Etest methods. In addition, rifampin + gentamicin and vancomycin + ampicillin/sulbactam resulted in synergy against SA 179 according to Etest methods. No antagonism was detected among both isolates. Time-kill experiments are listed in Figs. 1 and 2. Alone, all agents did not possess significant activity against both strains of SA 118 and SA 179, as they were tested at 0.5 MIC. For SA 118, the combinations that demonstrated additivity were gentamicin + daptomycin and gentamicin + quinupristin-dalfopristin. Gentamicin + ampicillin-sulbactam, gentamicin + vancomycin, and vancomycin + quinupristin/dalfopristin demonstrated synergy against SA 118 according to time-kill methods. Against SA 179, the combinations that demonstrated additivity were gentamicin + ampicillin-sulbactam, gentamicin + linezolid, gentamicin + quinupristin-dalfopristin,
Log10 CFU/ml
10
8
growth control vancomycin, ampicillin/sulbactam, quinupristin/dalfopristin, rifampin linezolid, gentamicin gentamicin + ampicillin/sulbactam
6
vancomycin + quinupristin/dalfopristin gentamicin + quinupristin/dalfopristin gentamicin + linezolid*
4
rifampin + gentamicin* gentamicin + vancomycin* rifampin + ampicillin/sulbactam*
2 0
8
16
24
Time (hour)
* = P < .05 Fig. 2. Select synergistic and additive time-kill experiments against SA179 GISA.
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and vancomycin+quinupristin-dalfopristin. In addition, gentamicin + vancomycin, rifampin + ampicillin/sulbactam, and rifampin + gentamicin resulted in synergy against SA 179 according to time-kill methods. When comparing both methods, agreement between Etest and time-kill methods for detecting antimicrobial synergy was 66.6% for hVISA and 71.4% for GISA.
susceptibility to vancomycin should be preformed to confirm these findings. Caution is warranted as additional investigations are necessary in humans before these results are applied to clinical practice.
Acknowledgments We would like to thank Anne Bolstrum and Irene Miloff.
4. Discussion In the face of mounting antimicrobial resistance, the need to evaluate new therapeutic options against S. aureus exist. Combination therapy, using agents that together achieve synergistic or additive activity is one potential means to optimize the pharmacokinetic and pharmacodynamic properties of new and existing antimicrobials. In the present investigation, we examined the potential effectiveness of numerous antimicrobial combinations against 2 S. aureus isolates from 1987 and 1989, which display heterogeneous resistance to vancomycin by utilizing Etest and time-kill methods to evaluate synergy. We were encouraged by the activity of a number of combinations that demonstrated either for synergistic or additive effects against hGISA and GISA. Among the 21 combinations that we tested against hGISA, numerous combinations with gentamicin significantly resulted in synergy or additivity. In addition, we evaluated the utility of the Etest method to detect antimicrobial synergy and compared these results to time-kill experiments. Although each method evaluates different end points, we found 66.6% to 71.4% agreement. These results are similar to a previously published comparison of the checkerboard, time-kill, and Etest methods (White et al., 1996). Other studies have found similar favorable agreement when comparing the Etest to time-kill methods. (Bonapace et al., 2002; Manno et al., 2003; Orhan et al., 2005; Pankey et al., 2002). The Etest method has significant advantages over traditional methods of synergy detection including being less labor-intensive and easier to perform, which may be of utility in the clinical microbiology laboratory. However, although it is practical method compared with time-kill experiments, the Etest method does not provide a quantitative measure of bacterial density in evaluating antimicrobial activity. In addition, compared with other traditional methods such as the checkerboard method, although both methods use the FIC index to evaluate synergy, Etest methods are not based on conventional microdilution MIC data, which use 2-fold dilutions. Therefore, it is important to take these considerations into account when interpreting synergy data generated from Etest methodology. Although we found good agreement between both methods, we recognize that depending on the methodology used, results often vary, which has been demonstrated in other studies (Bonapace et al., 2002; Cappelletty and Rybak, 1996; White et al., 1996). Further investigations in other S. aureus strains that display reduced
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